Leptospirosis is a zoonotic disease of global importance caused by spirochetes of the genus Leptospira. Case fatality rates are as high as 25%, with severe disease variably due to hepatic dysfunction, renal failure, and pulmonary hemorrhage in previously healthy people. The whole genome sequences of two Leptospira interrogans serovars, Lai and Copenhageni, have recently been determined. The aim of this R21 project is to use this sequence data to develop high-density microarrays that will be useful to investigators in the field for studying gene expression profiling and genomic composition of pathogenic Leptospira. This technology is critical for further developing the field of leptospiral microbiology, and will be applicable to experimental questions of pathogenicity, vaccine development, diagnostics and taxonomy. Specific aim 1 is to design, fabricate and validate high-density oligonucleotide arrays (500,000 features), based on the whole genome sequences of Leptospira interrogans serovars Copenhageni and Lai deposited into public databases. These microarrays will be composed of unique 25-mers that will represent virtually the entire sequenced 4.8 MB serovar Lai and Copenhageni Fiocruz L1-130 genomes. The microarray will be validated by hybridization with labeled genomic DMA from both Lai and Copenhageni, which will allow for testing of the ability of microarray hybridization to differentiate between closely related genomes whose sequences are already known. In Specific Aim 2, changes in genomic DNA composition, organization and gene expression profiles will be assessed by microarray analysis of the highly virulent L. interrogans serovar Copenhageni strain L1- 130 genome strain attenuated by serial passage in vitro. Large-scale changes will be associated with in vivo virulence as assessed by changes in LD50 and leptospiral tissue burden as determined by quantitative real time PCR. Gene expression profile differences will be determined under in vitro conditions that, at least in part, mimic conditions of in vivo growth. These experiments will provide the basis for future work examining mechanisms of leptospiral pathogenesis, determining potential vaccine candidates, developing more sensitive diagnostic tests and refining leptospiral taxonomy. [unreadable] [unreadable]